Single-nucleus and spatial transcriptome identify brain landscape of gene regulatory networks associated with behavioral maturation in honeybees

Animal behavior is associated with the gene regulatory network (GRN) coordinating gene expression levels in the brain. The eusocial honeybees with natural behavioral plasticity provide an ideal model for studying the relationship between brain activity and behaviors. Here, using single-nucleus RNA sequencing and spatial transcriptomics, we profile the expression changes of 121,247 brain cells associated with the behavioral maturation of honeybees from nursing to foraging outside. We identified 24 subpopulations of brain cells, including Kenyon, glia, optic lobes, and olfactory projection neurons. Compared to non-social insects, Drosophila, the Keyon cells display a high diversity in the mushroom body of honeybees. Integrating spatial and cellular profiles finds that the mushroom body and optic lobes show specifically high activities of the regulons led by different transcription factors. The stripe regulon is activated explicitly in the foragers’ small- and middle-type Keyon cells implicated in spatial learning and navigation behavior. These suggest that the activity of individual brain cells is coordinated by specific GRNs during the behavioral transition of honeybees. Our results provide a deeper understanding of the behavior-associated brain heterogeneity in GRN, which may be a key driver for the division of labor in social life.